Ajuwon et al., Afr J Tradit Complement Altern Med. (2016) 13(3):1-15

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1 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): HEPATIC, RENAL AND ENDOGENOUS ANTIOXIDANT STATUS MODULATORY EFFECTS OF CHRONIC CO-SUPPLEMENTATION OF ROOIBOS (ASPALATHUS LINEARIS) AND RED PALM OIL (ELAEIS GUINEENSIS) IN MALE WISTAR RATS Olwle Rzq Ajuwon 1,#, Oluwfemi Omoniyi Oguntieju 2, Jenine Lucst Mrnewick 1,* 1 Oxidtive Stress Reserch Centre, Institute of Biomedicl nd Microil Biotechnology, Fculty of Helth nd Wellness Sciences, Cpe Peninsul University of Technology, South Afric 2 Nutrition nd Chronic Diseses Reserch Unit, Deprtment of Biomedicl Sciences, Fculty of Helth nd Wellness Sciences, Cpe Peninsul University of Technology, South Afric # Present Address: Redox Lortory, Deprtment of Humn Biology, Fculty of Helth Sciences, University of Cpe Town Medicl School, Anzio Rod, Oservtory 7925, Cpe Town, South Afric. * Corresponding Author: Professor Jenine L. Mrnewick Oxidtive Stress Reserch Centre, Institute of Biomedicl nd Microil Biotechnology, Fculty of Helth nd Wellness Sciences, Cpe Peninsul University of Technology, South Afric. Emil: mrnewickj@cput.c.z Astrct Bckground: This study investigted the heptic, renl nd endogenous ntioxidnt sttus modultory effects of chronic cosupplementtion of rooios nd red plm oil (RPO) in mle Wistr rts. Mterils nd Methods: Rts were rndomized into four groups (n=10/group) nd fed dily either stndrd rt chow (SRC) nd wter (Group I), SRC nd the queous rooios extrct (2% w/v; Group II), SRC nd RPO (200 µl/dy) with wter (Group III), or SRC nd RPO (200 µl/dy) with rooios (2% w/v; Group IV) for 22 weeks. Results: Chronic feeding of rooios, RPO or their comintion did not induce ny dverse heptic or renl effects, s shown y serum levels of lkline phosphtse, lnine- nd sprtte minotrnsferse, lctte dehydrogense, lumin, cretinine, lood ure nitrogen nd uric cid. Histopthology nlyses showed tht liver from the three supplementtion groups displyed norml heptic historchitecture. Chronic feeding of RPO did not influence the redox sttus in the lood nd liver significntly. Supplementtion of rooios lone for 22 weeks significntly (P<0.05) incresed the reduced glutthione (GSH) level, GSH/GSSG rtio nd ctlse (CAT) ctivity in the liver. Co-supplementtion of rooios nd RPO significntly (P<0.05) incresed plsm totl polyphenol content, trolox equivlent ntioxidnt cpcity (TEAC), s well s lood GSH nd GSH/GSSG rtio. Plsm conjugted dienes (CD) nd heptic mlondildehyde (MDA) levels were reduced significntly y the comintion tretment, which lso incresed significntly the ctivity of CAT, glutthione reductse (GR), s well s the GSH/GSSG rtio in the liver. Conclusion: Our results suggest tht chronic feeding of n queous fermented rooios extrct lone or in comintion with RPO modulte the endogenous ntioxidnt system in rts. This modultion could e indictive of threshold effect nd/or n dditive effect indicting positive interction etween the ioctive components in the rooios nd RPO; however future studies re needed to elucidte the nture nd specific mechnisms involved. Also, the helth sttus of the individul/experimentl niml should e considered efore ntioxidnt supplementtion, s it is suggested to ffect the outcome. Key words: endogenous ntioxidnt system; glutthione redox sttus; lipid peroxidtion; rooios; red plm oil Introduction Oxidtive stress, the imlnce etween the production of free rdicls nd the nturl ntioxidnt ility of cells hs een estlished s either cuse or consequence of severl disese sttes (Dle-Donne et l., 2006; Khnsri et l., 2009; Lpps et l., 2011). Although, rective oxygen nd nitrogen species (RONS) re generlly considered s highly rective nd cytotoxic molecules, recent evidence is suggesting tht esides their noxious effects, they prticipte in importnt physiologicl processes such s defence ginst pthogens, signl trnsduction nd poptosis (Vlko et l., 2007). Rective oxygen nd nitrogen species, including superoxide rdicl nion (O2 ) nd hydroxyl (OH ) rdicls produced during oxidtive stress, re highly rective nd they cn ttck importnt mcromolecules within their vicinity, resulting in dmge to importnt cell structures, such s lipids nd memrnes, proteins nd nucleic cids (Dlle-Donne et l., 2006; Vlko et l., 2007). Lipid peroxidtion s mjor consequence of oxidtive stress-induced dmge my disrupt cellulr functions nd memrne integrity, leding to pthophysiologicl ltertions nd cell deth (Dlle-Donne et l., 2006; Shieh et l., 2010). Impirment of the ntioxidnt defence system of the cell, including inctivtion/inhiition of ntioxidnt enzymes, s well s n ltertion of the glutthione (GSH) redox sttus, tht is crucil for cellulr thiol-ntioxidnt defence system nd cellulr metolic mchinery, lso form prt of the consequences of oxidtive stress (Mlireddy et l., 2012). To prevent redox imlnce nd oxidtive dmge tht is consequent to oxidtive stress, living cells hve developed iologicl defence system, consisting of n rry of enzymtic nd non-enzymtic ntioxidnts, to prevent dmge due to oxidtive 1

2 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): stress. However, overproduction of RONS, with susequent oxidtive stress my overwhelm the endogenous ntioxidnt defence system, s is the cse during the onset or progression of disese stte. Therefore, dietry ntioxidnt therpy re seriously sought fter nd regrded s promising strtegy to strengthen the cellulr ntioxidnt defence system nd prevent oxidtive stress-medited cellulr injury. Disese prevention using whole plnt extrcts is gining scientific ttention worldwide, prtly ecuse the plnts re sources of ioctive phytochemicls, with ntioxidnt, disese prevention nd therpeutic properties (Mlireddy et l., 2012), nd lso ecuse epidemiologicl studies re providing convincing evidence tht diets rich in fruits, vegetles, tes nd spices re ssocited with lower risk of chronic diseses (Heer, 2013). Rooios is unique South Africn herl te produced from the leves nd stems of Asplthus lineris Dhlg (Leguminose). It is nturlly cffeine-free nd hs low tnnin content when compred to Cmelli sinensis tes, ut high in unique ntioxidnt polyphenols (Jouert et l., 2008). Rooios hs flvonoid profile tht is distinctly different from those found in Cmelli sinensis, including the β-dihydroxy-dihydrochlcone glucoside splthin, nd its cyclic counterprt spllinin, oth of which re unique to rooios (Re et l., 1994; Shimmur et l., 2006), the dihydrochlcone nothofgin, s well s flvonols including orietin, iso-orientin, vitexin, isovitexin, luteolin, quercetin nd chrysoeriol mong others (Re et l., 1994; Brmti et l., 2002; Jouert et l., 2008). Studies hs shown tht rooios exert heptoprotective effect in cute nd chronic liver dmge in rts (Ulicn et l., 2003; 2008), nd modulte oxidtive stress y preventing lipid peroxidtion nd improving glutthione redox sttus in rt tissues nd in humns (Nikolov et l., 2007; Mrnewick et l., 2011; Pntsi et l., 2011; Awoniyi et l., 2012). Red plm oil (RPO) is the edile oil otined from crude plm oil extrcted from the oil plm plnt, (Eleis guineesis), fter modified refining process, involving degumming nd leching, followed y de-cidifiction nd deodoristion y moleculr distilltion (Ngendrn et l., 200). It is n ntioxidnt-rich oil, with lmost equl proportions of sturted (mostly plmitic cid) nd unsturted (mostly oleic nd linoleic cid) ftty cids (Smnthmurthi et l., 2000). Red plm oil is mde up of cocktil of ft solule ntioxidnts, including vitmin E (70% s tocotrienols nd 30% s tocopherols), crotenoids (minly α- nd β-crotene), s well s lycopene, squlene nd coenzyme Q10 (Al-Sqer et l., 2004). The eneficil effects of RPO hs een demonstrted in vrious studies. Red plm oil hs een shown to e hypocholesterolemic (Wilson et l., 2005), protect herts sujected to ischemi/reperfusion injury (Esterhuyse et l., 2006) nd inhiit lipid peroxidtion nd modulte oxidtive stress in different models of oxidnt-medited injury (Nrng et l., 2005; Eriymremu et l., 2008; Budin et l., 2011). The oserved helth effects shown y mny medicinl plnts hve een ttriuted to the dditive or synergistic interction of the vrious phytochemicls they contin, rther thn to one single phytochemicl. Also, evidence exists to show tht polyphenols nd ntioxidnt vitmins my result in the synergistic modultion of lipid peroxidtion, oxidtive stress, nd co-oxidtion of dietry ntioxidnts (Gorelik et l., 2005; De Kok et l., 2008). Therefore, we hypothesized tht chronic feeding of rooios nd RPO together, my result in the synergy of their eneficil effects. Furthermore, most experimentl nd intervention studies on rooios nd RPO involved either cute or su-chronic feeding in rodent models of disese or in nimls with extensive pthology. This study investigted the heptic, renl nd responses of the endogenous ntioxidnt system in pprently helthy Wistr rts to chronic supplementtion of rooios nd RPO. Mterils nd Methods Red Plm Oil nd Aqueous Rooios Extrct Preprtions Fermented rooios extrct (RTE, 2% w/v) ws prepred y the ddition of freshly oiled tp wter to rooios leves nd stems t concentrtion of 2g/100 ml (Mrnewick et l 2003). The mixture ws llowed to stnd t room temperture for 30 minutes with constnt stirring, filtered nd dispensed into wter ottles. The queous rooios extrct ws fed to rts d liitum nd fresh rooios ws prepred every second dy. The RPO used in this study (Crotino TM king ft, Crotino SDN BHD, Johr-Bhru, Mlysi) ws fed to the rts orlly (200 µl, equivlent to 7g/kg diet) on dily sis in the morning, efore the nimls hd ccess to the stndrd rt chow. Animl Tretment nd Experimentl Design Forty pthogen-free, mle Wistr rts weighing 220 ± 24 g were used in this study. The nimls were otined nd housed t the Primte Unit of Stellenosch University (Tygererg Cmpus, South Afric). The rts were housed individully in stinless steel wired-ottom cges fitted with polypropylene houses under controlled environment, mintined t temperture of etween o C, with 12 h light drk cycle nd 50-54% humidity. The rts were fed stndrd rt chow (SRC) d liitum nd hd free ccess to tp wter or the queous rooios extrct. The nimls received humne cre in ccordnce with the Principle of Lortory Animl Cre of the Ntionl Medicl Reserch nd the Guide for the Cre nd Use of Lortory Animls of the Ntionl Acdemy of Sciences (Ntionl Institute of Helth Puliction no , revised 1978). The study protocol ws pproved y CPUT s Fculty of Helth nd Wellness Sciences Reserch Ethics Committee (Ethics Certificte no: CPUT/HAS-REC 2010/A003). After cclimtiztion in the experimentl niml holding fcility for 1 week, the rts were rndomized into four groups of 10 nimls ech, nd treted for 22 weeks s follows: Group I (control group): Fed SRC with ccess to wter s the sole source of drinking fluid for the durtion of the study. Group II (rooios group): Fed SRC with ccess to rooios (2% w/v) s the sole source of drinking fluid for the durtion of the study. Group III (red plm oil group): Fed SRC nd red plm oil [200 µl (equivlent to 7g/kg diet) per dy], with ccess to wter s only source of drinking fluid for the durtion of the study. Group IV (rooios + red plm oil group): Fed SRC nd red plm oil (200 µl per dy), with ccess to rooios (2% w/v) s the only source of drinking fluid for the durtion of the study. The generl conditions of the rts were monitored dily throughout the study nd ody weights recorded weekly nd t scrifice (end of 22 weeks). Fluid intke ws monitored t intervls of 2 dys throughout the study. At the end of the experimentl period, fter n overnight fst, nimls in ll the groups were scrificed under sodium pentoritl nesthesi (0.15 ml/100g ody weight, 2

3 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): i.p.). Approximtely 8 ml of lood ws collected vi the dominl ort nd liquoted into tues with (EDTA) nd without nticogulnt to otin plsm nd serum, respectively. Plsm/serum ws seprted immeditely y centrifugtion t g for 5 min t 4 o C. The liver ws removed, wshed twice with ice-cold PBS (10 mm phosphte uffered sline ph 7.2) to remove residul lood, lotted to dry, nd weighed. A slice of the liver smple ws tken nd fixed in 10% uffered formldehyde solution for histologicl exmintion. The remining liver tissue ws immeditely snp-frozen in liquid nitrogen nd stored t -80 o C for iochemicl nlysis. Histopthologicl Anlysis Histopthologic nlyses of the liver smples were performed t the Deprtment of Antomy nd Histology, Stellenosch University (Tygererg Cmpus, South Afric). Formlin-fixed liver tissues were wshed in tp wter, dehydrted in seril ethnol, clered in xylene nd emedded in prffin. A 3-5 µm thick section ws mde from the prffin-emedded lock nd stined with hemtoxylin nd eosin for microscopic ssessment. The slides were exmined under light microscopy y pthologist who ws lind to the protocol of the study. Solule Solids, Totl Polyphenols, Flvonol nd Flvnol Content of the Aqueous Rooios Extrct The solule solids content of the rooios extrct ws determined grvimetriclly (twelve repetitions) fter drying 1 ml liquots of the extrct t 70 o C for 24 hours. The totl polyphenol content of the queous rooios extrct ws determined using the Folin Cioclteu s phenol regent ccording to the method descried y Singleton et l. (1999) nd results expressed s mg gllic cid equivlents/mg solule solids. The flvnol content of the queous rooios extrct ws determined colorimetriclly t 640 nm using p- dimethylminocinnmldehyde (Treutter, 1989). Results were expressed s mg ctechin stndrd equivlents/mg solule solids. The flvonol/flvones content ws determined spectrophotometriclly t 360 nm nd results were expressed s mg quercetin stndrd equivlents/mg solule solids (Mzz et l., 1999). In Vitro Totl Antioxidnt Cpcity of Rooios Extrct nd Red Plm Oil Oxygen Rdicl Asornce Cpcity Assy This ssy mesures the ntioxidnt cpcity of plnt nd iologicl smples s rte of the peroxyl rdicl-generted decline in the fluorescence of fluorescein. The oxygen rdicl sornce cpcity (ORAC) of rooios extrct nd RPO ws determined ccording to method descried y Ou et l. (2001) with some modifictions. Briefly, 12 µl of diluted smple or trolox stndrd ws mixed with 138 µl of fluorescein (14 µm) nd 50 µl of AAPH (4.8 mm) dded, to initite the free rdicl ttck. Fluorescence (excittion 485, emission 538) ws recorded every 1 min for 2 hr in Fluoroskn Ascent plte reder (Thermo Fisher Scientific, Wlthm, Mss., USA). Results were expressed s µm Trolox equivlents (TE)/L or µm Trolox equivlents (TE)/g. Trolox Equivlent Antioxidnt Cpcity Assy The Trolox equivlent ntioxidnt cpcity (TEAC) of the queous rooios extrct nd RPO ws determined ccording to the method descried y Re et l. (1999). This method mesures the rdicl scvenging ility of ntioxidnts ginst ABTS +. The ABTS + solution ws prepred 24 h efore use y mixing ABTS slt (8 mm) with potssium peroxodisulfte (140 mm) nd then storing the solution in the drk until the ssy could e performed. The ABTS + solution ws diluted with distilled wter (1:20) to give n sornce of 1.50 t 734 nm. Briefly, 25 μl of smple or trolox stndrd ws mixed with 275 μl ABTS + solution in 96-well cler plte. The plte ws incuted for 30 min t room temperture nd the sornce red t 734 nm in Multiskn Spektrum plte reder (Thermo Fisher Scientific, Wlthm, Mss., USA). Results were expressed s μm TE/L or µm TE/g. Ferric Ion Reducing Antioxidnt Power Assy The ferric ion reducing ntioxidnt power (FRAP) of the rooios extrct nd RPO ws determined using the method descried y Benzie nd Strin (1996). The FRAP regent ws prepred y mixing cette uffer (300 mm, ph 3.6), TPTZ (10 mm in 100 mm HCl) nd FeCl3.6H2O (20 mm) in rtio of 10:1:1, v/v/v). Briefly, 10 μl of smple or scoric cid (AA) stndrd ws dded to 300 μl FRAP regent in 96-well cler plte. The plte ws incuted t room temperture for 30 min, nd sornce red t 593 nm in Multiskn Spektrum plte reder (Thermo Fisher Scientific, Wlthm, Mss., USA). Results were expressed s μmol scoric cid equivlent/litre (AAE/L) or µmol AAE/grm. High Performnce Liquid Chromtogrphy Anlysis of the Aqueous Rooios Extrct The queous rooios extrct ws filtered (Whtmn no 4) nd chromtogrphiclly seprted on n Agilent Technologies 1200 series HPLC system ccording to n dpted method descried y Brmti et l. (2002). The HPLC system consisted of G1315C diode rry nd multiple wvelengths detector, G1311A quternry pump, G1329A uto-smpler, nd G1322A degsser. A 5 µm YMC- Pck Pro C18 (150 mm x 4.6 mm i.d.) column ws used for seprtion nd cquisition ws set t 287 nm for splthin nd 360 nm for other components. The moile phses consisted of wter (A) contining 300 µl/l trifluorocetic cid nd methnol (B) contining 300 µl/l trifluorocetic cid. The grdient elution strted t 95% A, chnging to 75% A fter 5 min nd to 20% A fter 25 min nd ck to 95% A fter 28 min. The flow rte ws set t 0.8 ml/min, the injection volume ws 20 µl nd the column temperture ws set t 23 o C. Peks were identified sed on the retention time of the stndrds nd confirmed y comprison of the wvelength scn spectr (set etween 210 nm nd 400 nm). 3

4 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): High Performnce Liquid Chromtogrphy Anlysis of RPO Vitmin E content of RPO Vitmin E in RPO ws determined s isoforms of tocopherol nd tocotrienol. Extrction ws done y shking 1 g of RPO in 5 ml of solute ethnol for 30 min, followed y centrifugtion t 5000 g for 10 min. An liquot of the top vitmin E lyer (20 µl) ws injected into chromtogrphic system (Agilent Technology 1200 series), using n nlyticl column YMC-Pck Pro C18 (150 x 4.6 mm, i.d.) with the UV-visile wvelength detector set t 296 nm. The moile phse consist of A (cetonitrile:methnol:isopropnol:wter; 45:45:5:5, v/v) nd B (cetonitrile:methnol:isopropnol; 50:45:5, v/v) nd elution ws crried out t flow rte of 1mL/min. Moile phse A ws progrmmed to B within 10 min nd this condition mintined for nother 15 min efore returning to the originl condition. The content of tocopherols nd tocotrienols were quntified y compring the retention time nd/or pek re with stndrds (Iql et l., 2007). Crotenoids Content of RPO Crotenoids from RPO were extrcted with tetrhydrofurn:dichloromethne (1:1, v/v) nd nlysed on n Agilent Technology 1200 series HPLC with the visile detector set t 450 nm ccording to modified method of Rutench et l. (2010). Twenty microlitre of extrcted smples were injected utomticlly into the column (YMC-Pck Pro C30, 250 x 4.6 mm i.d., room temperture) nd isocrtic elution performed on moile phse consisting of methnol: cetone (9:1, v/v) with flow rte set t 1 ml/min. Peks were identified sed on the retention time of the α- nd β-crotene stndrds. Preprtion of Liver Homogentes The liver tissue ws homogenized on ice in 10 volumes of 50 mm phosphte uffer contining 1 mm EDTA nd 0.5% Triton- X (ph 7.5). The homogente ws trnsferred into tues nd centrifuged t g for 10 min t 4 o C. The superntnt ws collected, divided into liquots, nd stored t -80 o C until used for nlyses of ntioxidnt cpcities, lipid peroxidtion, ctivity of ntioxidnt enzymes nd glutthione sttus. Protein content of iologicl smples (erythrocytes nd liver homogente) ws determined using the BCA protein ssy kit supplied y Pierce (Illinois, USA). Antioxidnt Cpcity of Plsm nd Liver Smples To void protein interference in ntioxidnt cpcity ssys, su-smples of plsm nd liver homogentes were precipitted with 0.5 M perchloric cid (1:1, v/v) nd centrifuged t g for 10 min t 4 o C. Superntnts were collected s protein free frctions (Roles-Snchez et l., 2011). Plsm totl polypnenol, s well s ORAC, TEAC nd FRAP ssys (plsm nd liver) were performed s previously descried for the rooios extrct nd RPO (Benzie nd Strin, 1996; Re et l., 1999; Singleton et l., 1999; Ou et l., 2001). Liver nd Kidney Function Mrkers Serum levels of lnine trnsminse (ALT), sprtte trnsminse (AST), lkline phosphtse, lctte dehydrogense (LDH), lumin, totl protein, cretinine (CREA), lood ure nitrogen (BUN) nd uric cid were mesured on Medic EsyRA utomted clinicl chemistry nlyser (Medic Corportion Bedford, Mss., USA), using stndrd commercil kits (Medic Corportion Bedford, Mss., USA). Oxidtive Stress nd Antioxidnt Sttus Biomrkers Plsm nd Heptic Lipid Peroxidtion Lipid peroxidtion ws ssessed y mesurement of conjugted dienes (CD) nd mlondildehyde (MDA). Plsm nd liver MDA were ssyed s MDA-TBA dducts using HPLC with UV-visile detector ccording to Khoschsorur et l. (2000). Conjugted dienes were estimted ccording to the method of Reckngel nd Glende (1984). After the initil extrction of the lipid content from the plsm nd liver homogentes, the lipid residues were dissolved in cyclohexne nd CDs were mesured spectrophotometriclly t 234 nm nd results expressed s nmol/l or nmol/g tissue in plsm nd liver, respectively. Heptic nd Erythrocyte Antioxidnt Enzymes Activity Ctlse (CAT) ctivity in the erythrocytes nd liver homogentes were determined ccording to the method descried y Aei (1984), in which the rte of decomposition of hydrogen peroxide ws mesured t 240 nm. The ctivity of ctlse ws clculted using molr extinction coefficient of 43.6 M -1 cm -1 nd results expressed s µmole H2O2 consumed/min/mg protein. The ctivity of superoxide dismutse (SOD) ws determined ccording to the method of Crosti et l. (1987), nd SOD ctivity expressed s U/mg protein. Glutthione peroxidse (GPx) ctivity ws determined ccording to the method of Ellery nd Bredesen (2000). The ctivity of GPx ws clculted using the extinction coefficient (Ɛ) of 6.22 mm -1 cm -1 nd results expressed s nmol NADPH oxidized per min per mg protein. Glutthione reductse (GR) ws ssyed y method of Stl et l. (1969) nd result expressed s µmol NADPH oxidized per min per mg protein using the extinction coefficient (Ɛ) of 6.22 mm -1 cm -1. 4

5 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): Heptic nd Whole Blood Glutthione Redox Sttus The totl glutthione (GSH nd GSSG) ws mesured using Bioxytech GSH/GSSG-412TM commercil kit (OxisReserchTM, Portlnd, USA), sed on method descried y Tietze (1969). Aliquots of whole lood with 3 mm freshly prepred M2VP (for GSSG determintion) or without M2VP (for GSH determintion) were precipitted with 5% (w/v) metphosphoric cid (MPA), while liver smples were homogenized (1:10) in 15% (w/v) TCA contining 1 mm EDTA for GSH determintion nd in 6% (v/v) PCA contining freshly prepred 3 mm M2VP nd 1 mm EDTA for GSSG determintion on ice. After centrifugtion t g for 10 min, 50 µl of superntnt (from whole lood or liver homogente) ws dded to 50 µl of glutthione reductse (1U) nd 50µL of 0.3mM DTNB. The rection ws initited y ddition of 1 mm NADPH to finl volume of 200 µl. The chnge in sornce ws monitored t 410 nm for 5 min nd levels clculted using pure GSH nd GSSG s stndrds. GSH concentrtion ws clculted s the difference etween totl glutthione nd 2GSSG. Sttisticl Anlysis Vlues were expressed s men ± SD. Dt were tested for normlity nd differences etween groups mens were estimted using one-wy nlysis of vrince (ANOVA) followed y the Student-Newmn-Keuls test for ll pirwise comprisons. The Kruskl- Wllis test, non-prmetric nlogue to the one-wy ANOVA ws used to test for group differences when dt ws not normlly distriuted. Results were considered sttisticlly significnt t P<0.05, or mrginlly significnt t P<0.1. All the sttistics were crried out using MedClc v softwre (MedClc softwre v, Mrikerke, Belgium). Results Phytochemicl Content nd Antioxidnt Cpcity of Plnt Mterils The phytochemicl content nd in vitro ntioxidnt cpcity of the queous rooios extrct nd RPO re shown in Tle 1. The totl polyphenolic content of the rooios extrct is 1.06 ± 0.00 mg GAE/mL, 63% of which were mde up of flvonols nd flvnols. HPLC nlysis of the rooios extrct reveled the presence of peks consistent with ptterns showed y stndrds including splthin, iso-orientin, orientin, rutin/hyperoside nd others (chromtogrm not shown). Quntittively, the HPLC nlysis showed splthin (29.98 ± 0.08 µg/ml), iso-orientin (25.98 ± 0.52 µg/ml), orientin (18.61 ± 0.13µg/mL) nd hyperoside/rutin (14.55 ± 2.26µg/mL) were the min flvonoids in the queous rooios extrct (Tle 1). The totl polyphenolic content of RPO used is 0.17 ± 0.01 mg GAE/100g. The tocopherol content of the oil is 98µg/g, while the tocotrienol content is 386µg/g. The totl crotene content (α nd β) is 53 µg/g. The in vitro ntioxidnt cpcity of the RPO, mesured s ORAC, FRAP nd TEAC vlues re 175 ± µmol TE/100g, ± 0.28 µmol AAE/100g nd 0.16 ± 0.03 µmol TE/100g, respectively (tle 1). Tle 1: Phytochemicl constituents nd in vitro ntioxidnt cpcity of queous rooios extrct nd red plm oil Aqueous rooios extrct Red plm oil Constituents Concentrtion Constituents Concentrtion Solule solids (mg/ml) 2.85 ± 0.39 α-tocopherol (µg/g) ± 2.30 Totl polyphenol (mg GAE/mL) 1.06 ± 0.02 β/γ-tocopherol(µg/g) 6.20 ± 0.94 Flvonol (mg QE/ ml) 0.48 ± 0.01 δ-tocopherol (µg/g) ± 1.66 Flvnol (mg CE/mL) 0.19 ± 0.01 α-tocotrienol (µg/g) ± 1.52 Asplthin (µg/ml) ± 0.08 β/γ-tocotrienol (µg/g) ± 2.74 Orientin (µg/ml) ± 0.13 δ-tocotrienol (µg/g) ± 1.55 Iso-orientin (µg/ml) ± 0.52 α-crotene (µg/g) ± 1.16 Vitexin (µg/ml) 6.07 ± 1.05 β-crotene (µg/g) ± 2.92 Iso-vitexin (µg/ml) 7.18 ± 0.20 Totl polyphenol (mg GAE/100g) 0.17 ± 0.01 Hyperoside/rutin (µg/ml) ± 2.26 SFA (%) 51 Quercetin (µg/ml) 0.89 ± 0.18 MUFA (%) 38 Luteolin (µg/ml) 0.22 ± 0.06 PUFA (%) 11 Chrysoeriol (µg/ml) 0.25 ± 0.01 ORAC (µmol TE/100g) ± ORAC (µmol AAE/mL) 1.69 ± 0.03 FRAP (µmol AAE/100g) ± 0.28 FRAP (µmol TE/mL) 5.20 ± 0.04 TEAC (µmol TE/100g) 0.16 ± 0.03 TEAC (µmol TE/mL) 4.79 ± 0.33 Vlues re men ± SD. Solule solid is men of 12 determintions while other prmeters re men of 5 determintions. AAE (scoric cid equivlent), CE (ctechin equivlent), GAE (gllic cid equivlent), QE (quercetin equivlent), TE (trolox equivlent), SFA (sturted ftty cid), MUFA (mono-unsturted ftty cid), PUFA (poly-unsturted ftty cid). Dily Intke Profile of Rooios nd Red Plm Oil The dily phenolic, vitmin E nd crotene intkes of the vrious rt groups consuming the queous rooios nd RPO re shown in tle 2. These intkes were sed only on the rooios nd RPO intkes, nd do not tke into ccount the SRC intke. Dily wter intke etween the control group nd the group fed RPO lone ws similr. Rooios intke, totl phenolic, flvonol nd flvonol 5

6 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): intkes per dy etween the two groups tht were fed rooios were not significntly different. Also, the dily vitmin E nd crotene intkes etween the group fed RPO lone nd the group fed comintion of rooios nd RPO were lso similr. Tle 2: Dily phenolic, vitmin E nd crotenoid intkes of the vrious rt groups consuming the queous rooios extrct nd RPO Tretment Control RTE RPO RTE + RPO Wter/rooios intke/dy/100 g BW (ml) 6.63 ± ± ± ± 0.32 Totl phenolic intke (mg GAE/dy/100 g BW) ND 7.22 ± 0.33 ND 7.09 ± 0.34 Flvonol intke (mg QE/dy/100 g BW) ND 2.91 ± 0.13 ND 2.85 ± 0.14 Flvnol intke (mg CE/dy/100 g BW) ND 1.10 ± 0.05 ND 1.08 ± 0.05 Vitmin E intke/dy (µg/100g BW) ND ND ± ± 0.75 α-crotene intke/dy (µg/100g BW) ND ND 0.86 ± ± 0.04 β-crotene intke/dy (µg/100g BW) ND ND 1.06 ± ± 0.05 Clcultions of the totl phenolic, flvonol nd flvonol intkes were clculted sed on the solule solid intke otined from the verge rooios consumption per dy. Vlues re men ± SD (n=10). ND (not determined), BW (ody weight), CE (ctechin equivlent), GAE (gllic cid equivlent), QE (quercetin equivlent), RTE (queous rooios extrct), RPO (red plm oil). Body nd Liver Weight Chnge The effect of chronic feeding of rooios, RPO or their comintion on ody weight gin, liver weight nd reltive liver weight is shown ove in tle 3. Chronic feeding of rooios, RPO or their comintion for 22 weeks, did not hve ny deleterious effect on the ody weight gins cross ll the groups, however, consuming these plnt products lone or in comintion did significntly (P<0.05) decrese the solute liver weight nd the reltive liver weight of the rts when compred to the control nimls. Liver nd Kidney Function Mrkers nd Histopthology Tle 3 lso shows the effect of chronic feeding of rooios nd RPO on mrkers of liver nd kidney functions. The serum levels of hepto-specific enzymes (ALP, ALT, AST nd LDH) remined similr to tht of the control, fter feeding with the rooios extrct, RPO or their comintion for 22 weeks. Dt from the histopthologicl exmintion of liver tissues showed tht none of the feeding regimens hd deleterious effect on the liver, with the liver tissues showing norml historchitecture (figure 1). Also the levels of ALB, BUN, CREA nd uric cid which cn e used to ssess dmge to the kidney, ws unffected y chronic feeding of the rooios extrct, RPO or their comintion for 22 weeks. Tle 3: Effect of chronic feeding of queous rooios extrct, RPO or their comintion on weight chnges nd mrkers of liver nd kidney functions. Tretment Control Rooios RPO Rooios + RPO Body weight (g) ± ± ± ± Liver weight (g) ± ± 1.87 * ± 1.37 * ± 1.48 * Reltive liver weight (%) 2.81 ± ± 0.32 * 2.39 ± 0.16 * 2.42 ± 0.17 * ALB (g/l) ± ± ± ± 1.10 ALP (U/L) ± ± ± ± ALT (U/L) ± ± ± ± AST (U/L) ± ± ± ± LDH (U/L) ± ± ± ± CREA (µmol/l) ± ± ± ± 1.47 BUN (mmol/l) 5.94 ± ± ± ± 0.53 Uric Acid (µmol/l) ± ± ± ± Vlues re men ± SD (n=8-10). *Significntly different from control t p<0.05. ALB (lumin), ALP (lkline phosphtse), ALT (lnine minotrnsferse), AST (sprtte minotrnsferse), BUN (lood ure nitrogen), CREA (cretinine), LDH (lctte dehydrogense). Plsm nd Heptic Antioxidnt Cpcity nd Lipid Peroxidtion Effect of chronic feeding of queous rooios extrct, RPO or their comintion on the ntioxidnt cpcity in the plsm nd liver is presented in tle 4. In the plsm, totl polyphenol nd TEAC sttus were unffected y chronic feeding of the queous rooios extrct or RPO when compred with control. However, comined feeding of the rooios extrct nd RPO significntly (P<0.05) incresed the totl polyphenol content nd TEAC sttus compred to control nimls. The ORAC nd FRAP sttus of the rts remined similr (P>0.05) to those of the control group fter eing fed the queous rooios extrct, RPO or their comintion for 22 weeks. In the liver, the ORAC, TEAC nd FRAP sttus in the groups chroniclly fed the queous rooios extrct, RPO or their comintion ws not sttisticlly (P>0.05) different when compred to the control group. Lipid peroxidtion ws ssessed s conjugted dienes (CD) nd mlondildehyde (MDA) levels. Plsm CD remined unchnged (P>0.05) compred to control rts when queous rooios extrct lone ws supplemented. Plsm CD level in the rts consuming RPO either lone or comined with rooios ws significntly (P<0.05) reduced when compred to control rts (tle 4). Supplementtion of rooios, RPO nd their comintion did not hve ny effect on plsm MDA s the MDA level ws similr cross ll tretment groups. In the liver, the level of CD ws not ffected y chronic 6

7 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): supplementtion of the fermented rooios extrct, RPO nd their comintion. Heptic MDA ws lso not ffected y chronic supplementtion of the fermented rooios extrct or RPO lone, however, when oth extrct were given together, heptic MDA ws significntly (P<0.05) lowered when compred to the control group (tle 4). c d Figure 1: Hemtoxylin nd eosin stined liver sections showing the effect of chronic feeding of queous rooios nd red plm oil on liver historchitecture. () control group, (, c nd d) rooios, red plm oil nd rooios + red plm oil group respectively, showing norml historchitecture of the liver. Tle 4: Effect of chronic supplementtion of rooios, red plm oil nd their comintion on plsm nd liver ntioxidnt cpcity nd lipid peroxidtion Tissue Prmeter Control RTE RPO RTE + RPO TP (µmol ± ± ± ± 4.39 GAE/L) TEAC (µmol ± ± ± ± TE/L) Plsm ORAC (µmol ± ± ± ± TE/L) FRAP (µmol ± ± ± ± AAE/L) CD (nmol/l) ± ± ± ± 5.28 MDA (µmol/l) 1.56 ± ± ± ± 0.33 TEAC (µmol ± ± ± ± 4.46 TE/g) Liver ORAC ((µmol ± ± ± ± 1.43 TE/g) FRAP (µmol 2.85 ± ± ± ± 0.39 AAE/g) CD (nmol/l) ± ± ± ± 1.17 MDA (µmol/l) ± ± ± ± 1.13 Vlues re men ± SD (n=10). Vlues in the sme row with different superscript re significntly different from ech other t p<0.05. CD (conjugted dienes), FRAP (ferric reducing ility of plsm), MDA (mlondildehyde), ORAC (oxygen rdicl sornce cpcity), TEAC (trolox equivlent ntioxidnt cpcity), TP (totl polyphenol), RTE (fermented rooios extrct), RPO (red plm oil), AAE (scoric cid equivlent), GAE (gllic cid equivlent), TE (trolox equivlent). 7

8 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): CAT (µmol H /min/mg protein) SOD (U/mg protein) c d GPx (nmol NADPH/min/mg protein) GR (µmol/nadph/min/mg protein) Figure 2: Effect of chronic supplementtion of rooios, red plm oil nd their comintion on erythrocyte () ctlse (CAT), () superoxide dismutse (SOD), (c) glutthione peroxidse (GPx) nd (d) glutthione reductse (GR). Brs represent men ± SD (n=9-10). Brs with different letters re significntly different from ech other t P<0.05. RTE (fermented rooios extrct), RPO (red plm oil). 8

9 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): CAT (µmol H 2 O 2 /min/mg protein) SOD (U/mg protein) c d GPx (µmol NADPH/min/mg protein) GR (µmol/nadph/min/mg protein) Figure 3: Effect of chronic supplementtion of rooios, red plm oil nd their comintion on heptic () ctlse (CAT), () superoxide dismutse (SOD), (c) glutthione peroxidse (GPx) nd (d) glutthione reductse (GR). Brs represent men ± SD (n=9-10). Brs with different letters re significntly different from ech other t P<0.05. RTE (fermented rooios extrct), RPO (red plm oil) 9

10 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): Erythrocyte nd Heptic Antioxidnt Enzymes Fig 2 nd 3 shows the results of the effect of chronic feeding of rooios nd RPO on erythrocyte nd heptic ntioxidnt enzymes. In the erythrocyte, the ctivities of SOD, GPx nd GR remined unchnged s result of chronic feeding of the queous rooios extrct, RPO or their comintion, when compred to the control group (figures 2, 2c & 2d). The ctivity of CAT in rts chroniclly fed the rooios extrct or RPO lone ws similr to those of control rts. However, comined feeding of the rooios extrct nd RPO significntly (P<0.05) incresed the erythrocyte CAT ctivity when compred to the control group (Fig 2). Heptic SOD nd GPx remin unchnged cross ll tretment groups when compred to control group (figures 3 & 3c). Feeding the fermented rooios extrct lone or comined with RPO significntly (P<0.05) incresed the CAT ctivity when compred with the control group (figure 3). GR ctivity ws not ffected y supplementtion of fermented rooios or RPO lone. However when the two plnt extrct were supplemented together, the ctivity of GR ws significntly (P<0.05) incresed compred to the control group (figure 3d). Whole Blood nd Heptic Glutthione Sttus Tle 5 shows the glutthione sttus cross ll experimentl groups. In the lood, supplementtion of the fermented rooios extrct lone, or together with RPO for 22 weeks, significntly (P<0.05) incresed the level of reduced glutthione (GSH) when compred to the control. Rts tht were fed RPO lone for 22 weeks hd GSH vlues tht were comprle to those of the control. Oxidized glutthione (GSSG) ws not ffected y chronic supplementtion of fermented rooios extrct, RPO or their comintion s GSSG level remin similr in ll the tretment groups compred with the control group. The GSH/GSSG rtio ws incresed significntly in rts supplemented with fermented rooios extrct either lone or in comintion with RPO, when compred with control rts. In the liver, supplementtion of fermented rooios extrct, RPO or their comintion for 22 weeks did not ffect GSH level when compred to control. Chronic supplementtion of fermented rooios extrct lone or together with RPO significntly (P<0.05) decresed the liver GSSG levels nd significntly incresed the GSH/GSSG rtio when compred to the control. Tle 5: Effect of chronic supplementtion on rooios, red plm oil nd their comintion on whole lood nd heptic glutthione redox sttus in Wistr rts Tissue Prmeter Control RTE RPO RTE + RPO GSH (µmol/l) ± ± ± ± Whole lood GSSG (µmol/l) ± ± ± ± 3.00 GSH/GSSG ± 2.85 c ± ± 4.01 c ± 4.90 GSH (µmol/g) 9.10 ± ± ± ± 1.30 Liver GSSG (µmol/g) 0.35 ± ± ± ± 0.06 GSH/GSSG ± ± ± ± 3.34 Vlues re men ± SD (n=10). Vlues in the sme row with different superscript re significntly different from ech other t p<0.05. GSH (reduced glutthione), GSSG (oxidized glutthione), RTE (fermented rooios extrct), RPO (red plm oil). Discussion The sensitive lnce etween the pro-oxidnt nd ntioxidnt systems in the ody is of utmost importnce when determining the stte of helth, well-eing nd survivl of orgnisms. A loss in the functionl ctivity of the ntioxidnt systems, s result of overproduction of rective free rdicls or depletion of the ntioxidnt molecules, might result in the disruption of the pro-oxidnt to ntioxidnt rtio, creting stte of oxidtive stress, which hs een implicted s either cuse or consequence of mny disese sttes (Dlle-Donne et l., 2006). The fct tht oxidtive stress hs een implicted in the pthophysiology of most disese sttes suggest tht ugmenting the cellulr ntioxidnt defence system my e promising nd prgmtic pproch to prevent or slow down the progression of such diseses. Epidemiologicl findings nd experimentl dt from niml nd humn studies hs shown tht diets rich in plntderived foods, contining high levels of nturl ntioxidnts, my contriute to reduced mortlity from diseses (Heer, 2004; 2013), thus ttention is now centred on dietry phytochemicls not only s n effective intervention in disese onset nd progression, ut lso s n intervention for sustining nd promoting overll helth. Fruits, vegetles, herl tes nd spices re rich sources of dietry phytochemicls, which individully or in comintion my enefit helth. These enefits re ttriuted to the presence of essentil dietry micronutrients, fires, ntioxidnt vitmins, trce elements, nd polyphenolic compounds, mostly flvonoids (Yhi, 2010). In this study, the effects of sustined feeding of two ntioxidnt-rich nturl plnt products on the endogenous ntioxidnt system in mle Wistr rts were investigted. Specificlly, the ctivities of ntioxidnt enzymes nd the glutthione redox sttus were mesured in the liver nd lood of these rts. Prior to the feeding experiment, the phytochemicl composition s well s the in vitro ntioxidnt cpcity of the queous rooios extrct nd red plm oil were quntified in order to determine whether the phenolic nd ntioxidnt content of the plnt extrcts plyed n importnt role in their oserved helth effects. In ccordnce with previously pulished studies, HPLC quntifiction of the rooios extrct showed tht splthin is the mjor flvonoid present in rooios, long with others such s iso-orientin, orientin, vitexin, iso-vitexin nd hyperoside/rutin (Re et l., 1994; Brmti et l., 2002; Shimmur et l., 2006). Different isoforms of vitmin E, α- nd β-crotene were lso quntified in the RPO used in this study. Literture on whether consumption of polyphenol-rich diet will led to n increse in plsm totl polyphenol level nd plsm totl ntioxidnt cpcity (TAC) hs een inconclusive, with some studies reporting n increse (Leontowicz et l., 2003; Gorinstein et l., 2006), while others hve reported no increse (Ali et l., 2003; Grci-Solis et l., 2008; Kim et l., 2008). It hs een 10

11 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): suggested tht since ssys for ntioxidnt cpcity lck specificity, single mesurement of TAC is insufficient (Hlliwell, 2009). Therefore mesuring TAC using ttery of ssy methods is recommended. Thus, for the current study, we mesured the plsm nd heptic ntioxidnt cpcity using three well known TAC (ORAC, FRAP nd TEAC) ssys. Our results showed tht chronic consumption of fermented rooios extrct or RPO for 22 weeks did not increse the plsm totl polyphenol or plsm nd liver TAC mesured s ORAC, FRAP or TEAC. However, when the rooios extrct nd RPO were fed together, the comintion resulted in significnt increse in plsm totl polyphenol nd plsm TAC mesured only s TEAC. Some studies in humns hve shown tht the plsm totl polyphenol content prllels the plsm TAC fter the intke of polyphenol-rich diets (Henning et l., 2004; Torin et l., 2009; Wng et l., 2012), suggesting tht phenolic compounds re the mjor contriutors to the plsm TAC, fct tht we lso suspected my e true in nimls. The TEAC ssy is sed on the inhiition of the sornce of the ABTS rdicl ction (ABTS + ) y ntioxidnts. The ABTS + rdicl cn e soluilised in oth queous nd orgnic medi, llowing the ssy to mesure lipophilic ntioxidnts like crotenes nd tocopherols (Krdg et l., 2009) tht re found in RPO. Bsed on this, we cn ssume tht the TEAC ssy is more sensitive nd more pplicle to our study, since we supplemented with plnt extrcts tht re rich in oth hydrophilic nd lipophilic ntioxidnts. The fct tht the comined supplementtion resulted in n increse in plsm totl polyphenol levels nd TAC mesured s TEAC, showed tht there my e positive interction in the ility of oth extrcts to increse the plsm polyphenol nd TAC nd tht there my e threshold tht hs to e reched efore significnt increse is evident. Results from this study showed tht chronic supplementtion of the queous rooios extrct, RPO or their comintion for 22 weeks did not hve ny deleterious effects on the growth of the rts, s ody weight gins remined similr cross ll tretment groups. However, ll the rts in the tretment groups mintined lower solute- nd reltive liver weights compred to the control rts, nd no cler reson could e offered to explin this. Furthermore, no mortlity ws recorded throughout the 22 weeks of supplementtion nd fluid intkes in ll the supplementtion groups were similr to those of the control group. A widely used mesure of sfety of medicinl nd plnt extrcts in literture is to determine their effect on serum levels of liver nd kidney function mrkers. Incresed serum levels of lnine minotrnsferse (ALT), sprtte minotrnsferse (AST), lkline phosphtse (ALP) nd lctte dehydrogense (LDH) re used s surrogte mrkers for heptic injury. In this study, we oserved tht chronic feeding of queous rooios extrct, RPO or their comintion for 22 weeks resulted in no significnt chnges in the serum levels of ALP, ALT, AST nd LDH nd tht it ws similr to those of the control nimls. The sme trend ws lso oserved in the serum levels of kidney function mrkers (lumin, cretinine, lood ure nitrogen, nd uric cid), tht were lso similr to those of the control group consuming wter. Therefore, our result showed tht chronic feeding of rooios; RPO or their comintion for 22 weeks did not exhiit dverse heptic or renl effects. The result of the effect of rooios, RPO or their comintion on the liver ws further corroorted y the results of the histopthologicl exmintion of the liver tissues, which showed tht liver tissues from ll the supplementtion groups exhiited norml historchitecture, similr to those of the control. This oservtion from our study ecomes ll the more importnt, especilly with recent cses of possile dverse heptic effects of rooios. Sinislo et l. (2010) reported cse of 42-yer-old ptient dignosed with low grde B-cell mlignncy, Wldenstrom s mcrogloulinemi, six yers erlier, who temporrily experienced elevted liver enzymes fter consuming reltively lrge mount of rooios. The study however re-ffirms the excellent sfety record of rooios nd concluded tht contmintion of the rooios herl te y some heptotoxic compound, genetic predisposition nd/or disesed stte of the ptient my e responsile for the effect. A recent study lso reported cse of cute heptitis nd liver filure ssocited with dily ingestion of rooios nd uchu herl te in 52-yer-old mn with history of hyperlipidemi nd stge III chronic kidney disese secondry to IgA nephropthy (Engels et l., 2013). Previous studies hve reported on the sfety of rooios showing tht it is heptoprotective nd does not influence liver functions negtively in nimls (Ulicn et l., 2003; 2008; Mrnewick et l., 2003) nd humns (Mrnewick et l., 2011), fct tht ws confirmed y the current study. Agin these results drw ttention to the helth-sttus of the individul eing very importnt when considering supplementtion with dietry ntioxidnts. Incresed ROS genertion during oxidtive stress my result in lipid peroxidtion, which cuses considerle ltertion in the functions nd structurl orgniztion of the cell memrne. Since lipid peroxidtion is well-known mechnism of toxicity exhiited y mny xenoiotics, it is expected tht ntioxidnt supplementtion will ugment the cell s endogenous ntioxidnt defence system, nd therefore inhiit lipid peroxidtion. Results from this study showed tht in the plsm, chronic feeding of RPO lone or together with the queous rooios extrct significntly lowered the level of CDs while the MDA level ws not ffected y ny of the tretment. In the liver, CD levels were lso unffected y either chronic supplementtion of rooios extrct, RPO or their comintion. Heptic MDA did not respond to chronic feeding of either queous rooios extrct or RPO, ut the comined feeding significntly lowered the MDA level, showing tht the comintion of the extrcts elicits protective ntioxidnt effect, pointer to positive interction of the two extrcts. The min endogenous enzymtic ntioxidnts re CAT, SOD, GPx nd GR. These enzymes hve different cellulr locliztion nd re responsile for reduction of different rective oxygen species. SOD ctlyses the dis-muttion of superoxide rdicls to oxygen nd hydrogen peroxide (H2O2) which is degrded y CAT to oxygen nd wter. GPx nd GR re importnt enzymes of the glutthione defense system. GPx protects memrne lipids, protein nd nucleic cids y eing responsile for removl of wide rnge of peroxides, rnging from orgnic hydro-peroxides to H2O2 using GSH s co-sustrte while GR regenertes GSH from GSSG t the expense of NADPH. Determintion of the ctivity of ntioxidnt enzymes s mrker of protective role of chronic feeding of the rooios extrct, RPO or their comintion, did not show ny mjor effect in our study. In the erythrocytes, we oserved significnt increse only in the ctivity of CAT s result of comined feeding of the rooios extrct nd RPO, while the other three enzymes (SOD, GPx nd GR) remined unchnged y ny of the feeding protocols. In the liver, SOD nd GPx ctivities were not ffected y ny of the tretments; CAT ctivity ws incresed y chronic feeding of the rooios extrct lone or in comintion with RPO while the comintion tretment lso incresed the GR ctivity. Evidence from literture hs shown tht the effect of the consumption of polyphenol- nd other ntioxidnt-rich diets on the ctivity of ntioxidnt enzymes remin contrdictory. Some uthors hve reported no increse fter consumption of polyphenol-rich diet contining red wine, green te, s well s fruits nd vegetles in humns (Vn der Gg et l., 2000; Vn den Berg et l., 2001; Young et l., 2002), while others hve reported n increse in erythrocyte GPx nd GR fter consumption of grpe skin extrct diet for one week (Young et l., 2000). Rt feeding experiments reveled tht, green te leves cused n increse in SOD nd CAT ctivity (Lin et l., 1998), thyme oil nd thymol prevented ge-induced decline in GPx nd SOD in 11

12 Ajuwon et l., Afr J Trdit Complement Altern Med. (2016) 13(3): geing rt rin (Youdim nd Dens, 2000), lycopene fed to young femle rts incresed erythrocyte ctivity of SOD, GPx nd GR (Breinholt et l., 2000), while gvge of nturl flvonoids to femle rts decresed the ctivity of CAT, GPx nd GR (Breinholt et l., 1999). These studies show tht the responses of the ntioxidnt enzymes do not follow set ptterns, ut could e diet-, tissue- nd species-specific (Crwford et l., 2000). Reduced glutthione (GSH) is the mjor nd most importnt intrcellulr thiol ntioxidnt in living orgnisms nd plys importnt roles in myrid of cellulr functions, such s mintennce of reduced protein thiols nd coordinting cellulr ntioxidnt defence, including detoxifiction of hydrogen- nd lipid peroxides, s well s direct non-enzymtic scvenging of free rdicls, such s hydroxyl nd superoxide rdicls. Mesuring the GSH level or its oxidized form (GSSG) is universlly ccepted method of detecting oxidtive stress nd the GSH/GSSG rtio cn serve s good indictor of systemic oxidtive sttus nd disese risk. Results from this study showed tht while lood GSSG remin similr cross ll tretment groups, chronic feeding of rooios lone or in comintion with RPO for 22 weeks significntly incresed the GSH level in the lood, nd consequently GSH/GSSG rtio ws lso significntly incresed in the rooios lone nd comintion group. Feeding RPO lone did not show ny significnt effect on either GSH level or the GSH/GSSG rtio. The incresed GSH level my e scried to the ility of flvonoids in rooios to increse GSH synthesis y upregulting the mrna expression of γ-glutmylcysteine synthetse (γ-gcs), the rte limiting enzyme in the GSH iosynthetic pthwy, since previous studies hve shown tht polyphenolic compounds from plnts incresed the γ-gcs ctivity nd GSH contents (Jeon et l., 2003; Chen et l., 2004; Moskug et l., 2005) nd the rooios extrct hs significntly higher totl polyphenol content when compred to the RPO in the current study. In the liver, feeding rooios, RPO or their comintion for 22 weeks did not show ny effect on the GSH level, however GSSG level ws significntly reduced in the rooios lone or the comintion group resulting in n incresed GSH/GSSG rtio in this groups. This oservtion my e s result of the ility of the polyphenolic sustnces in rooios to directly scvenge free rdicls nd prevent oxidtion of GSH to GSSG therey improving the redox sttus of the heptic cells. In conclusion, the present results suggest tht chronic feeding of RPO lone to rts for 22 weeks neither induced ny heptic or renl toxic effect nor influence the ntioxidnt/oxidnt lnce (redox sttus) in the lood nd liver of the rts significntly. Our results lso show tht chronic feeding of rooios extrct lone for 22 weeks did not induce ny dverse heptic or renl effect, ut modulte the ntioxidnt/oxidnt lnce y incresing CAT, GSH (liver) nd GSH/GSSG rtio (lood nd liver). This study showed tht feeding rooios nd RPO together for 22 weeks did not induce ny deleterious effect on the liver nd kidney, the comintion tretment however improved the glutthione redox sttus (GSH nd GSH/GSSG) in the lood nd liver of the rts. We scried these oservtions to the polyphenolic compounds present in rooios, since the mgnitude of these effects ws similr to wht ws otined when rooios ws fed lone nd RPO lone ws ineffective. Also feeding rooios nd RPO together significntly incresed plsm totl polyphenol nd ntioxidnt cpcity mesured s TEAC nd decresed heptic MDA even when rooios or RPO were ineffective on this iomrker, however ecuse of the mgnitude of these effects, we could not scrie the comine effects to synergistic interction etween the wter-solule ioctive compounds in rooios nd the lipid solule components in RPO. It is pertinent to sy tht since this study ws conducted in pprently helthy rts, future studies should explore supplementtion of these extrcts in rts under extensive pthology or those tht re pre- or post- exposed to n oxidtive stress inducer to fully understnd the mechnisms ehind these oserved effects. Acknowledgements Fermented rooios (superior grde) plnt mteril used in the study ws generous gift from Mr Arend Redelinghuys, Rooios Limited, Clnwillim, South Afric. RPO used is gift from Prof Jcques Vn Rooyen. The uthors grtefully cknowledged the stffs of the Primte Unit, Stellenosch University (Tygererg Cmpus), South Afric for cre nd the mintennce of the nimls, nd Mr Fnie Rutench for technicl ssistnce. Funding from the Cpe Peninsul University of Technology is grciously cknowledged. Conflict of Interest nd Funding: The uthors declre no competing finncil interest. References Aei, H. (1984). Ctlse in vitro. Methods Enzymol. 105: Ali, M., Horcjo, C., Brvo, L. nd Goy, L. (2003). Effect of grpe ntioxidnt dietry fier on the totl ntioxidnt cpcity nd the ctivity of liver ntioxidnt enzymes in rts. Nutr. Res. 23: Al-Sqer JM, Sidhu JS, Al-Hooti SN, Al-Amiri HA, Al-Othmn A, Al-Hji L, Ahmed, N., Mnsour, I.B. nd Minl, J. (2004). Developing functionl foods using red plm olein. IV. Tocopherols nd tocotrienols. Food Chem. 85: Awoniyi, D.O., Aou, Y.G., Mrnewick, J. nd Brooks, N. (2012). 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